Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies

Lindsay R Ditzler; Arundhuti Sen; Michael J Gannon; Amnon Kohen; Alexei V Tivanski

doi:10.1021/ja205409v

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Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies

Journal article

Peer reviewed

Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies

Lindsay R Ditzler, Arundhuti Sen, Michael J Gannon, Amnon Kohen and Alexei V Tivanski

Journal of the American Chemical Society, Vol.133(34), pp.13284-13287

08/31/2011

DOI: 10.1021/ja205409v

PMCID: PMC4343314

PMID: 21809877

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http://doi.org/10.1021/ja205409vView

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Abstract

Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.

Gold - chemistry

Enzymes, Immobilized - metabolism

Escherichia coli - enzymology

Methotrexate - metabolism

Surface Properties

Microscopy, Atomic Force - methods

Molecular Dynamics Simulation

Tetrahydrofolate Dehydrogenase - metabolism

Details

Title: Subtitle: Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies
Creators: Lindsay R Ditzler - The Department of Chemistry, The University of Iowa, Iowa City, Iowa 52245, United States
Arundhuti Sen
Michael J Gannon
Amnon Kohen
Alexei V Tivanski
Resource Type: Journal article
Publication Details: Journal of the American Chemical Society, Vol.133(34), pp.13284-13287
DOI: 10.1021/ja205409v
PMID: 21809877
PMCID: PMC4343314
NLM abbreviation: J Am Chem Soc
ISSN: 1520-5126
eISSN: 1520-5126
Publisher: United States
Grant note: R01 GM065368 / NIGMS NIH HHS
Language: English
Date published: 08/31/2011
Academic Unit: Chemistry
Record Identifier: 9983985916402771

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